In the coming year, we will continue to examine the effects of the adenine nucleotide interactions with placental estradiol dehydrogenase. The 8-azido and 8-bromo derivatives of adenine nucleotides will be used for affinity labeling of the enzyme, followed by studies of the modified enzymes. Also, the reduced forms of (4-pro-S-3H)-8-azidonicotinamide adenosine dinucleotide and its (4-pro-R-3H) isomer will be synthesized and used as an affinity label. Studies will follow to determine if the covalenty bound cofactor is suitably positioned to serve as a cofactor in the dehydrogenase reaction and if the 4-pro-R or 4-pro-S hydrogen is transfered to the steroid substrates. We will continue our studies on the isolation and characterization of "active" metabolites of estrogens and their bound adducts. After adduct identification, various cell culture experiments will be performed to demonstrate that such transformations can occur at the cellular level. Further protein biochemistry, including two-dimensional gel electrophoresis, solubilization, and purification of microsomal proteins, will be undertaken to examine the extent and significance of the covalent binding of estrogen to protein.